Although several cationic polymers have been developed in recent years for siRNA delivery,25 none have been FDA approved and there still exists a void in the mechanistic details of the delivery process due to the lack of extensive structure/function studies, such as those done with DNA. While both siRNA and DNA have the same charge density, siRNA is distinctly different from DNA in its ability to be condensed by polycations due to its rigid-rod like nature. This difference greatly affects the extracellular and intracellular stability of siRNA- nanoparticles, two features that are critical for efficient delivery. To this end, I propose the design and use of a new type of delivery polymer that encapsulates siRNA along with a new disassembly-specific probe to study the kinetics of siRNA nanoparticle disassembly within the cytoplasm. To facilitate the study of intracellular nanoparticle disassembly, a delivery vector that condenses siRNAs into a nanoparticle is required because naked siRNA on its own cannot cross the cellular membrane. Towards this goal, I have designed a cationic polymer via thiol-ene chemistry that condenses siRNAs into 50-60 nm particles. To ensure that this new class of polymers will be biodegradable, able to get into cells, and non-toxic, I will investigate polymer degradation via 1H NMR, nanoparticle surface charge via zeta potential, and the degree of polymer and nanoparticle cytotoxicity in a human cervical cancer (HeLa) cell line via the MTT cytotoxicity assay. For the disassembly kinetics study, the siRNA to be encapsulated within the polyplex will be labeled with an infra-red fluorescent indocyanine green (ICG) dye. ICG's fluorescence is concentration sensitive due to an aggregation induced self- quenching mechanism. The labeled siRNA-ICG probe should function as follows: strong repulsion between siRNA molecules due to their strong negative charges should prevent siRNA-ICG aggregation and thus promote fluorescence. However, upon condensation with cationic polymers to form polyplexes, the accumulation of several siRNA-ICG molecules in a 50-60 nm particle due to strong charge interactions should initiate aggregation induced self-quenching of ICG leading to a sharp decrease in its fluorescence intensity. Thus, the dependence of ICG's fluorescence on nanoparticle disassembly should provide an on/ff indication of nanoparticle stability. The detection of intracellular disassembly via this siRNA-ICG probe along with a mathematical model will form the basis for quantitatively determining the kinetics of siRNA release. After developing and testing this probe with the newly designed polymer, I will create new model library of degradable polymers via thiol-ene chemistry and correlate the disassembly kinetic parameters obtained from the mathematical model with transfection efficiencies to provide new insights into the contributing role of nanoparticle disassembly in siRNA delivery. Information gathered from this study will also be used to develop new structure-function correlations that will guide the design of future polymer libraries towards accelerating the discovery of potent siRNA delivery vectors for RNAi cancer therapeutics. PUBLIC HEALTH RELEVANCE: My research plan involves a mechanistic study of siRNA delivery via a newly designed probe and polymer library in order to improve the design and thus accelerate the discovery of potent siRNA therapeutics. These potent therapeutic vectors will help realize the great potential of RNAi therapy in the treatment of human diseases such as cancer via silencing of cancer causing genes, thus advancing the goals of the NIH by improving human health through disease treatment.